PROJECT SUMMARY Failure to increase insulin secretion and reduce insulin clearance to overcome tissue insulin resistance leads to the development of type 2 diabetes (T2D). Components of the insulin axis (insulin sensitivity, insulin secretion, insulin clearance) are critical to the genesis of T2D, yet the factors that account for their dysfunction and their interactions with other factors are not understood. The nutritional components of diet pass through the intestinal barrier in a complex interaction with the gut microbiota (the microbiome) to impact glucose homeostasis. Our hypothesis is that change in three insulin axis traits is associated with gut microbial composition and function, and this association is modified by dietary components (e.g., whole grains, red meat) including systemic short chain fatty acids produced by the gut microbiota. This study will assess the insulin axis, the gut microbiota and diet in a cohort of 500 non-diabetic adults (half African American, half non- Hispanic White) over 2.5 years (sampled at three time points). Specific Aim 1 will administer, at each clinic visit, a 75-g oral glucose challenge with 0, 30, 60, 120 min measurements (of insulin, glucose, and C-peptide) to determine insulin sensitivity, secretion, and clearance; habitual diet will be determined by use of a validated Food Frequency Questionnaire. Specific Aim 2 will characterize the gut microbiome for each participant by performing 16S rDNA sequencing and low-pass metagenomic sequencing on stool samples collected at all three visits. Together, these data will test the hypothesis that increased insulin resistance, impaired insulin secretion, and decreased insulin clearance (all diabetogenic changes) developing over time are associated with a reduced (at baseline) or declining (over time) abundance of short chain fatty acid-producing bacteria in the gut, in part attributable to unhealthy dietary patterns. Specific Aim 3 will utilize samples collected at the three time points to probe the functional profile of the gut microbiome by conducting deep metagenomic sequencing and assessment of circulating short chain fatty acid levels in a subset of 180 individuals with extreme changes (increase and decrease) versus those with no change in insulin axis traits, thereby identifying microbial functions that underlie change versus stability in insulin axis traits. This study has high impact, yielding knowledge that can lead to novel microbiome-based diagnostics, prevention, and/or treatment measures (e.g., specific diets; antibiotic or probiotic treatment) to reduce the public health burden of T2D.